Abstract: 【Objective】 The HB (Homeobox) family of transcription factors plays a crucial role in regulating plant organ development and responding to both abiotic and biotic stresses. This study aims to elucidate the expression patterns and regulatory mechanisms of HB gene family members in Pyrus sinkiangensis during the overwintering process, providing a theoretical foundation for further exploration of the biological functions of the HB gene family in this species. 【Methods】 Based on the whole-genome database of Pyrus sinkiangensis, bioinformatics tools were utilized to identify members of the HB gene family. Comprehensive analyses were performed, including phylogenetic relationships, chromosomal localization, gene structure, promoter cis-acting elements, and family-wide collinearity. In addition, differential expression patterns of these genes during the overwintering process were examined using transcriptome data. 【Results】 A total of 93 HB gene family members were identified in the Pyrus sinkiangensis genome through bioinformatics approaches. Analysis of the physicochemical properties of these HB proteins revealed that their lengths range from 176 to 1196 amino acids, with isoelectric points (pI) ranging from 4.69 to 9.32. More than 75% of these proteins have a pI below 7.0, suggesting that the HB genes in Pyrus sinkiangensis likely encode acidic proteins. The molecular weight of these proteins ranged from 20.18 kDa to 135.08 kDa, with PsHB69 being the largest and PsHB74 the smallest. Among all members, only PsHB5 contains a signal peptide, while the remaining members lack signal peptides, indicating that the majority are not secreted proteins. Subcellular localization analysis showed that six members (PsHB90, PsHB54, PsHB15, PsHB48, PsHB36, and PsHB12) are localized in the chloroplast, while the others are localized in the nucleus. Chromosomal localization analysis revealed that the 93 HB gene family members are distributed across 17 chromosomes (Chr01-Chr17) in Pyrus sinkiangensis. On chromosome 15, the highest number of HB members is distributed, totaling 12, while the other chromosomes contain between 2 to 9 members each. Additionally, two pairs of closely located members on chromosomes 9 (PsHB46 and PsHB47) and 17 (PsHB92 and PsHB93) exhibit high sequence similarity. All four belong to the HD-ZIP IV subfamily, suggesting that these pairs may have resulted from tandem duplication events. Phylogenetic analysis indicated that the HB gene family in Pyrus sinkiangensis, along with Arabidopsis and Malus domestica, can be divided into eight subfamilies based on the classification of the Arabidopsis HB gene family and the HD-ZIP I-IV gene families in Malus domestica. Among these subfamilies, HD-ZIP I and HD-ZIP IV contain the most members, with a total of 34 genes. In Pyrus sinkiangensis, all HB members contain exons, with the number ranging from 2 to 20. Each HB gene in Pyrus sinkiangensis contains a coding sequence (CDS) region, although 59 members lack untranslated regions (UTRs). Conserved domain analysis of these proteins revealed that all members possess the HD domain, and a total of 25 types of domains were identified among the 93 members. Besides the HD domain, the Homeodomain-associated Leucine Zipper (HALZ) domain was the most abundant, which is thought to mediate protein-protein interactions. Collinearity analysis of the HB gene family in Pyrus sinkiangensis revealed 70 pairs of collinear genes. Interestingly, PsHB genes on chromosomes 8 and 15 appear as tandem duplicates but belong to different subfamilies. Further examination of the Ka/Ks ratio of these duplicated genes revealed values ranging from 0.06 to 0.49. Among the 70 pairs of collinear genes in Pyrus sinkiangensis, 67 pairs had a Ka/Ks ratio of less than 1, indicating that these genes are under purifying selection and that their sequences have been conserved throughout evolution. A cis-acting element analysis of the 2000 bp upstream promoter regions of the Pyrus sinkiangensis PsHB gene family members identified 12 types of cis-elements related to plant hormone and stress responses. Among these, 78.49% of the PsHB genes contain an abscisic acid-responsive element (ABRE), and 50.54% contain gibberellin-responsive elements (P-box and GARE-motif). This suggests that the HB gene family may play a role in mitigating environmental stress through hormone-mediated pathways. Additionally, 41.94% and 54.84% of the PsHB genes contain low-temperature responsive elements (LTR) and drought-responsive elements (MBS), respectively. Transcriptome data analysis during three stages of the overwintering process in Pyrus sinkiangensis revealed that 39 out of the 93 PsHB genes were differentially expressed. Of these differentially expressed genes, more than half (23 genes) showed peak expression during the coldest period in December (TM), 11 genes had the highest expression at the end of the overwintering period in March (TF), and the remaining 5 genes exhibited the highest expression at the beginning of overwintering in October. Notably, during the coldest period in December, PsHB11 and PsHB78 were upregulated by 10.5-fold and 7.0-fold, respectively, indicating that these two genes may play a significant role during bud dormancy. Approximately 42% of the HB genes were significantly differentially expressed during the overwintering period, with the majority reaching their peak expression in December, the coldest month, suggesting that the PsHB gene family plays a critical role in cold stress resistance. 【Conclusion】 The expression patterns of the 93 PsHB gene family members varied across different stages of the overwintering process. These findings provide new insights into the functional roles and regulatory mechanisms of HB genes in response to cold stress, laying a foundation for genetic improvement and environmental adaptability research in Pyrus sinkiangensis.
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